The Herpesviridae are the most prevalent virus family in humans, and are are a major source of morbidity and mortality. Their success is largely due to their ability to establish latent infections which are refractory to clearance by the immune system and antiviral therapy. Latency was defined many years ago, yet mechanisms involved in its establishment, maintenance, and reactivation remain obscure. The complexities of in vivo models have made it challenging to unravel specific aspects ofthe virus-host interplay that result in latency. Our understanding ofthe molecular nature of neuronal latency, as exhibited by herpes simplex virus (HSV), has been hampered by lack of a tractable in vitro system. The role of adaptive immunity in latency has been extensively studied, but the role of innate immunity is less clear. The roles played by pathogen recognition receptors (PRRs), cell-intrinsic immune effectors, and autophagy have not been studied in the context of latency and reactivation, but there is much evidence to suggest that these factors are critical for the establishment of latency. This evidence in presented in this proposal. Our overarching hypothesis is, therefore, that molecular sensing of virus infection, and the subsequent rapid development of innate immune responses are essential to dampen viral replication during the entry into latency. All aims will use the Gene Expression and Mouse Cores, and will be performed in collaboration with the Coen and Knipe Labs. In our first aim, we will test this hypothesis with a new in vitro HSV latency model in combination with genetically altered mice lacking components ofthe innate immune response. We will measure viral gene expression, viral chromatin structure, and activation of innate immunity. Our second aim, will utilize and create recombinant viruses lacking the ability to counter the innate immune response in our in vitro latency model. Our final aim will examine the role of the cellular autophagy pathway in the establishment and maintenance of latency. These will provide a synergistic approach to address this hypothesis, and advance our understanding of latency. If our hypotheses are correct, we will identify a pivotal role for innate immunity in latency, pointing toward new approaches for chemotherapeutic intervention for HSV. RELEVANCE (See instructions); Herpes simplex virus infections are a widespread source of disease worldwide. Infection of humans at any point in their lives leads to lifelong latent infection for which there is no vaccine or cure. This proposal will lead to improved knowledge of herpes simplex latent infection, and will open avenues for interfering with the transmission of, and disease caused by, this highly prevalent pathogen.